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Tip60 Might Be a Candidate for the Acetylation of Hepatic Carbonic Anhydrase I and III in Mice

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Tip60 Might Be a Candidate for the Acetylation of Hepatic Carbonic Anhydrase I and III in Mice Tip60 Might Be a Candidate For The Acetylation of Hepatic Carbonic Anhydrase I and III in Mice Nurdan GONUL BALTACI Ataturk University: Ataturk Universitesi Enver Fehim KOCPINAR Muş Alparslan Üniversitesi: Mus Alparslan Universitesi Harun Budak ( [email protected] ) Ataturk University https://orcid.org/0000-0002-7371-8959 Research Article Keywords: Carbonic anhydrases, Tip60, acetylation, circadian rhythm, mice Posted Date: March 22nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-323673/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Abstract Carbonic anhydrases (CAs) play an important role in maintaining pH balance by catalyzing the conversion of carbon dioxide to bicarbonate. Since this pH balance is critical to health, all organisms must develop mechanisms to control and regulate it. Although there is a great deal of literature on the biochemical, functional, and structural properties of the CA family, there is no enough knowledge on the regulation of CAs at gene and protein levels, especially their epigenetic regulation. In this study, impact of Tip60, a member of histone acetyltransferases family, on the expression of Ca1 and Ca3 genes in liver tissue was investigated at different zeitgeber time points in control and liver-specic Tip60 knockout mice (mutant) groups. First of all, Tip60 was specically knocked out in mouse liver the using Cre/loxP system and knockout rate was shown as 83% - 88% by southern blot. Expression proles of Ca1 and Ca3 genes in both groups were determined by Real-Time PCR at six different time points. While Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression prole was observed at ZT0 and ZT20. Hepatic Ca1 showed a robust circadian expression. While hepatic Ca3 showed almost the same level of expression at different time units. The expression of Ca1 and Ca3 signicantly decreased in the absence of Tip60 in mouse liver all time period. In conclusion, it was suggested for the rst time that Tip60 may be considered a candidate protein in the regulation of Ca1 and Ca3 genes, possibly by acetylation. Introduction Carbonic anhydrases (CAs), zinc-containing metalloenzymes that are commonly found in living organism, catalyze the reversible hydration of CO2 to bicarbonate (HCO-3) and proton (H+) [1-3]. Although these enzyme were rst discovered in hemolyzed blood, today it is known to have activity in many tissues such as kidney, liver, brain, muscle, and bone tissues [4-7]. CAs found in vertebrates (α-CA) are composed of 16 different isoenzymes and play a role in a wide variety of functions such as respiration, acid-base homeostasis, ion transport, bone resorption, taste preferences, ureagenesis, and gluconeogenesis [8-10]. Eight of these proteins are in the cytosol (CAI, CAII, CAIII, CAVII, CA VIII, CA X, CA XI and CA XIII), ve are transmembrane or membrane bound (CAIV, IX, XII, XIV and XV), two are in mitochondria (CAVA and VB), and one (CAVI) is secreted [11]. While there is a great deal of literature on the biochemical, functional, and structural properties of the CA family, there is no enough knowledge on the regulation of CAs at gene and protein levels, especially their epigenetic regulation [12]. Epigenetic mechanisms such as acetylation, phosphorylation, methylation, ubiquitination, sumoylation, and glycosylation are required to regulate gene expression and chromatin structure in mammalian cells without modulating the DNA sequence [13, 14]. Protein acetylation, which refers to the covalent binding of an acetyl group to an amino acid residue of a protein, is the most well-known along with phosphorylation [15]. The correlation between increased transcription and histone acetylation has been known for many years. Thus, acetylation regulates a number of metabolic and physiological processes by affecting protein functions, protein-DNA and protein-protein interactions, and subcellular localization of the protein [16, 17]. A recent study reported that members of the α-CA family, CAI (at the N-terminus), CAII (at the N-terminus, K18, K39, and K113), CAIII (at the N-terminus and K126), and CAXII (at K194), are acetylated by acetyltransferase enzymes. But Page 2/15 the proteins involved in this acetylation are still unknown [12]. TIP60 protein, a member of the histone acetyltransferases (HATs) protein family, has important and vital functions such as transcriptional regulations, DNA repair, cell cycle, apoptosis mechanism, cancer, circadian system and generation of cellular signals, both directly and indirectly. [18-21]. Studies have shown that the Tip60 protein is associated with many transcription factors and proto-oncogenes such as androgen receptor, c-Myb, c- Myc, STAT3, NF-Kb, E2F1, p53 and acts as a regulator / correlator [22-24]. The study was performed by Chen et al. showed that c-Myb transcription factor increases Ca1 expression by binding to its promoter in mouse erythroleukemia cells [25]. However, c-Myb inactivation is required to inhibit the Ca1 gene when the division of the cell is achieved. The cell goes to cancer without this suppression. It is also known that Tip60 is a regulating factor for c-Myb [22]. In this study, it is aimed that does Tip60 have a role in the regulation of hepatic Ca1 and Ca3 which are predominantly expressed in the liver? For this purpose, liver- specic Tip60 knockout mice was generated by using Cre/loxP recombination. Quantitative expression of Ca1 and Ca3 genes at different zeitgeber time (ZT) points were determined for both control and knockout groups and then compared each other. Ca7, which is not regulated by acetylation, was used as a negative control [12] Materials And Methods 2.1. Liver-Specic Conditional Knockout Mouse Model To generate liver-specic Tip60 knockout mice (mutant), Tip60 oxed mice (10-12 week old male) with loxP sites anking exons 1 and 9 of the Tip60 gene [21] were crossed to a SACre driver mouse line resulting in Cre-mediated deletion of Tip60 in the liver [26]. Mice were previously backcrossed to a C57BL/6N background for at least 10 generations. To delete Tip60, 10-12 week-old male mice (Tip60/- ; SA+/Cre-ERT2) were injected daily with tamoxifen (10mg/ml stock solution, Sigma, St. Louis, MO, USA) in corn oil for ve consecutive days. The control group (Tip60/;SA+/+), in which corn oil were injected. Genotyping was performed with gene-specic primers [21, 26]. Liver and kidney tissues were collected ve days after the last injection. Genomic DNA was isolated from both tissues and analyzed by southern blot. 2.2. Southern Blot Analysis Genomic DNA from liver and kidney tissues was isolated with the DNeasy Tissue kit (Qiagen Inc., Valencia, CA, USA) and digested with BamHI (NEB, Ipswich, MA, USA). DNA was separated on a 0.6% agarose gel and transferred onto Hybond-XL positive charged nylon membrane (GE Healthcare/Amersham Biosciences, Sweden). The membranes were hybridized with a 32P-dCTP labeled radioactive double-stranded DNA probe was prepared by random priming using an appropriate commercial kit according to manufacturer’s instructions (Amersham Rediprime™ II DNA Labeling System, GE Healthcare) and puried with the illustra ProbeQuant™ G-50 Micro Columns (GE Healthcare). Hybridization of the radioactive probe (100 µl) to the membrane was performed at 65°C overnight in the presence of a hybridization buffer. Membranes were washed with 2xSSC/0.1% SDS, 1xSSC/0.1% SDS, Page 3/15 and 0.1xSSC/0.1% SDS, at 60°C until the excess label was removed and exposed to a sensitive X-ray lm (Kodak X-Omat 1000,1000A ve 1000J Processors). 2.3. Experimental Animals, Feeding, and Zeitgeber Time At least 3 weeks prior any experiment, all mice were singly housed with food and water ad libitum under a 12‐hour‐light/12‐hour‐dark cycle (350 lux). Throughout this study, time is indicated using zeitgeber time (ZT) as the indicator for the phase of the rhythm, wherein ZT0 refers to the time that lights went on (06:00), and ZT12 refers to the time that the lights went off (18:00). ZT4, ZT8, ZT16, and ZT20 in this study are equivalent to 10:00, 14:00, 22:00, and 02:00 respectively [27]. Articial light was provided daily from ZT0 (06:00), with temperature (24 ± 1) °C, and humidity (55 ± 5%) kept constant [28]. In the rst set of experiments, 10–12‐week‐old male C57BL/6N mice were used and split up into six groups corresponding to the six chosen timepoints (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20). For the second set of experiments, we used 10–12‐week‐old male mutant mice (Tip60/- ; SA+/Cre-ERT2) and their respective control littermates [29]. 2.4. RNA Extraction and First Strand cDNA Synthesis Total RNA isolation from approximately 50 mg mice liver tissues was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen-74804) following the manufacturer’s instructions. Concentrations and purities of RNAs were measured by spectrophotometer. (Thermo Scientic, Multiskan GO, USA), RNA quality was checked by agarose gel electrophoresis and stored at −80°C until use. Total RNA was converted into rst strand cDNA using SuperScript III First-Strand cDNA kit system (Invitrogen, California, USA), utilizing random hexamers, according to the manufacturer's protocol. The resulting cDNAs were diluted to 100 ng/ μL with nuclease-free water and stored at −20 °C [30]. 2.5. Primers and Probes Design Primer3 software (v. 0.4.0) (http://bioinfo.ut.ee/primer3-0.4.0/) was used for gene-specic primers and probes design that met the following criteria; amplicon size 75-200 bp, ≤3 G or C repetitions, GC content 50-65%, ≤4 base repetitions, melting temperature (Tm) 60°C. Primers and probes were veried with Blast Tool (NCBI) to conrm its specicity for the desired target.
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